User:Sean P Corum/Notebook/PHIX174 Cell Free/2012/09/25

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m (Entry title)
m (Characterization B-C: Expression of PHIX174 promoters/UTRs fused to PX-UTR1-deGFP and PX-UTRX-deGFP.)
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* Cell-free expression 80%:
* Cell-free expression 80%:
** 30 μL extract (12May11)
** 30 μL extract (12May11)
-
** 12.1 μL water
+
** 3.1 μL water
 +
** 9 μL 100 μM malachite green (10 μM final)
** 0.9 μL 100 mM Mg-glutamate (1 mM final)
** 0.9 μL 100 mM Mg-glutamate (1 mM final)
** 2 μL 3 M K-glutamate (66.7 mM final)
** 2 μL 3 M K-glutamate (66.7 mM final)
Line 25: Line 26:
*# 1 μL 50 nM / 3^0 * 20% = 10 nM
*# 1 μL 50 nM / 3^0 * 20% = 10 nM
* Plate reader:
* Plate reader:
 +
*# 29 °C
 +
*# Shake: fast double orbital 30s
 +
*# Read: 625/655 nM @ gain = 175 w/ settings
 +
*# Read: 625/655 nM @ gain = 200 w/ settings
 +
*# Read: 625/655 nM @ gain = 225 w/ settings
 +
*# Loop: 2-5 every 3 m for 12 h
 +
** settings = full plate, high lamp energy, endpoint, bottom reading, other settings default

Revision as of 14:28, 25 September 2012

PHIX174 Cell Free Expression Main project page
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Characterization B-C: Expression of PHIX174 promoters/UTRs fused to PX-UTR1-deGFP and PX-UTRX-deGFP.

  • Quantifluore of pBEST-Pr-MG-apt-UTR1-deGFP-T500 = nM.
  • Do cell-free expression in batch-mode to characterize pBEST-Pr-MG-apt-UTR1-deGFP-T500. Idea is to start at lowish gain (say 50), and then increase gain at ~3-4 hr when expression is peaking until an 80% of max reading (80,000 counts of 100,000 max for the PMT) is achieved. This will then be the fixed gain for MGapt experiments.
  • Cell-free expression 80%:
    • 30 μL extract (12May11)
    • 3.1 μL water
    • 9 μL 100 μM malachite green (10 μM final)
    • 0.9 μL 100 mM Mg-glutamate (1 mM final)
    • 2 μL 3 M K-glutamate (66.7 mM final)
    • 22.5 μL 6 mM amino acids mix (1.5 mM final)
    • 4.5 μL PEG8000 40% (2% final)
  • Aliquot 6 × 9 μL into 394-well plate and then add plasmid (pBEST-Pr-MGapt-UTR1-deGFP-T500) range:
    1. 1 μL water = 0 nM
    2. 1 μL 50 nM / 3^4 * 20% = .123 nM
    3. 1 μL 50 nM / 3^3 * 20% = .370 nM
    4. 1 μL 50 nM / 3^2 * 20% = 1.11 nM
    5. 1 μL 50 nM / 3^1 * 20% = 3.33 nM
    6. 1 μL 50 nM / 3^0 * 20% = 10 nM
  • Plate reader:
    1. 29 °C
    2. Shake: fast double orbital 30s
    3. Read: 625/655 nM @ gain = 175 w/ settings
    4. Read: 625/655 nM @ gain = 200 w/ settings
    5. Read: 625/655 nM @ gain = 225 w/ settings
    6. Loop: 2-5 every 3 m for 12 h
    • settings = full plate, high lamp energy, endpoint, bottom reading, other settings default
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